Journal: PLoS ONE
Article Title: Viral Double-Strand RNA-Binding Proteins Can Enhance Innate Immune Signaling by Toll-Like Receptor 3
Figure Lengend Snippet: The 1b HCV polymerase can enhance IL6 production by BEAS-2B cells. A) Enhanced IL6 production in the presence of 1bΔ21 can be knocked down by SiRNA to TLR3. siRNAs specific to TLR3 (siTLR3), RIG-I (siRIG-I), or a nonspecific control (nsRNA) were transfected into at a concentration of 30 nM into cells 48 h prior to the addition of 1bΔ21 (0.15 µM) and poly(I:C) (0.13 µg/ml) to the culture medium. IL6 in the medium was collected 24 h later and quantified by ELISA. The data from 3 to 4 sets of samples are presented as a percentage of the IL6 in the sample treated with nsRNA (100%). B) IL6 production by BEAS-2B cells in response to stimulation by the single-stranded polyinosinic acid (0.13 µg/ml), poly(I:C) (0.13 µg/ml) or lipolysaccharide (LPS) (1 µg/ml), the agonist for TLR4 in the absence (Ø) or presence of 1bΔ21 (0.15 µM). C) A model of the 1b HCV polymerase that illustrates the location of the Δ1 loop (in light blue), the GDD active site (in yellow), and the short helix deleted in mutant m26–30 (in red). D) The 1bΔ21 polymerase and 1b.m26–30 can form a complex with the double-stranded S4 RNA. The S4 dsRNA was labeled with α- 32 P-CTP and used in an electrophoretic mobility shift assay. The gel image shown is from a non-denaturing stacked polyacrylamide gel of 5 and 20%. E) Effects of mutations in 1bΔ21 on IL6 production induced by poly(I:C). f denotes a reaction with no added proteins. Proteins 1bΔ21, 1b.m26–30, and the active site mutant GDA were added to the culture media (final concentrations 0.01, 0.1 or 0.5 µM) in the absence or presence of 0.13 µg/ml poly(I:C). LL37 was added to a final concentration of 3 µM. F) The 1b HCV polymerase (1bΔ21, 0.1 µM), but not the 2a polymerase (2aΔ21, 0.1 µM), allowed TLR3 to produce cytokines in response to viral dsRNAs. In addition to poly(I:C), the RNA ligands used were those extracted from Reovirus virions (ReoV), from the endornavirus BPEV, and the annealed transcripts of the sense and antisense strands made from the S4 cDNA of Reovirus (S4), and the transcript of the HCV 2a JFH-1.
Article Snippet: RdRp assays RdRp assays were performed as 20 µL reactions containing 20 mM sodium glutamate (pH 8.2), 12.5 mM DTT, 4 mM MgCl2 , 1 mM MnCl2 , 0.5% Triton X-100, 0.2 mM GTP, 0.1 mM ATP and UTP, 250 nM [α-32 P] CTP (MP Biomedicals), 2 pmol PE46 and 1 pmol LE19p as templates .
Techniques: Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis, Labeling, Electrophoretic Mobility Shift Assay